The overall goal of this project is to understand oxygen and peroxide activation and the molecular mechanisms by which the free energy released in the reduction of these substrates is couple to biologically useful chemistry in cytochrome oxidases and prostaglandin H synthases. Ns and ps time-resolved resonance Raman spectroscopy, time-resolved optical spectroscopy, low-temperature FTIR spectroscopy, and electron magnetic- resonance techniques will be the principal analytical tools. Recent technical advances to Michigan State with these spectroscopies will allow us to use them to study both mutant and wild-type cytochrome oxidase and prostaglandin synthase. With cytochrome oxidase, we intend to characterize pathways for proton conduction in and out of the active site that are relevant to both 02 reduction and proton pumping; specific residues that are potentially involved in ligand exchange processes at the metal centers will be targeted and studied in terms of their functions in the catalytic cycle. In these experiments, we intend to elucidate the means by which proton control of the dioxygen activation and reduction chemistry that occurs in cytochrome oxidase is implemented and the means by which the free energy released by electron transfer to reactive oxygen intermediates is used to drive proton translocation. For prostaglandin synthase, the reduction of peroxides will be studied with time-resolved techniques to understand the reaction sequence and the coupling to tyrosyl radical formation. Subsequent mutation analysis will target residues implicated by the crystal structure as being essential to the catalysis.